Non-invasive prenatal testing (NIPT)

Blanca Rodríguez Estrada
Genetics Lab Manager

Genetics PGS / PGT-A, Miscarriages and RPL

From this video you will find out:
  • What does prenatal screening test for?
  • How early in pregnancy can you test for chromosomal abnormalities?
  • What is noninvasive prenatal testing (NIPT) and what disorders can it screen for?
  • When is the best time to do the NIPT test?
  • What is cell-free DNA and why we can use it to do NIPT?
  • How does Single-Nucleotide Polymorphism (SNP) based NIPT work?
  • What is a normal fetal fraction?
  • How accurate is NIPT?
  • What are the disadvantages of the NIPT test?

What does NIPT test for and how accurate are results?

In this session, Blanca Rodríguez Estrada, Genetics Lab Manager at iGLS, in-house Reproductive Genetics Laboratory of the IVF Life Group, talked about the NIPT test, what is for and how accurate the results are.

Blanca Rodríguez Estrada started by defying prenatal testing, which is the determination of a pathological condition or illness in the embryo after implantation of the fetus or pregnant woman before delivery. Prenatal testing offers better management of pregnancy. It helps with better management of pregnancy thanks to risk adapt monitoring, sometimes the effects of conditions such as cardiovascular or digestive malformations may be solved by insulting treatment and lastly, in cases when fatal or the mother’s life is compromised, pregnancy termination.

Noninvasive prenatal testing (NIPT)

How to know the risk of pregnancy? First, it is important to pay attention to the patient’s medical history, to check if the patient had previous miscarriages and if so whether these miscarriages have been studied and whether this patient has a relative that suffers from a genetic condition. Moreover, maternal age has been proven to be related to an increase in embryo aneuploidies. Imaging techniques help clinicians give them an approach to fetal phenotype. There are also biochemical factors that can be markers of normal or abnormal pregnancies. Lastly, the sample collection of fetal material and its analysis is the most feasible approach, but also the most invasive.

The majority of pregnancies that are lost before delivery were carrying aneuploidy, which means they had an abnormal number of chromosomes, which is 46 XY or 46 XX. The graph shown presented the prevalence of chromosome aneuploidies detected during pregnancy, it shows that more than 50% are due to trisomy of chromosome 21, which causes Down syndrome, followed by trisomy of chromosome 18. Plus, a high number of miscarriages are caused by Turner syndrome, which is 45, X0.

Microdeletion syndromes have a major impact on fetal development, they are caused by small fragments of DNA being lost during cell division. The most common microdeletion syndromes include DiGeorge, Prader-Willi, etc. Even though those are the most common ones, they are not that common in the population, for example, DiGeorge syndrome is present in 1 of 4 000 to 6 4000 pregnancies.

The first-trimester screening is based on a maternal blood test and an echography. Within the maternal blood test, 2 main hormones are normally tested, Beta -Hcg (Human chorionic gonadotropin) and PAPP-A ((Pregnancy-associated plasma protein-A). However, it is known that other hormones, such as Alpha-Fetoprotein (AFP) or PIGF (Placental growth factor) have been included in some countries as co-markers. On the other hand, echography is a helpful tool to follow up on pregnancy. In this case, some markers can be measured to help the screening like body length measuring, the length between the head and the end of the column nasal bone, and the presence or absence of nasal bone and its length. Then the neutral translucency measures the fluid area between the skin and soft parts in cervical bones that it’s increasing in trisomies of chromosomes 13, 18 and 21.

The first-trimester screening measures at least 2 hormones, Beta -Hcg and PAPP-A and the nuchal translucency. This allows us to calculate the risk at weeks 11 to 13 of pregnancy. The results determine the classification of risk groups. These groups may change between countries because every health system has its own calculations based on incidents. For example, in Spain, patients are divided into 4 groups depending on the risk. The first group is of low risk where there is no need for an additional test, then there is a medium group that can be offered to perform a NIPT test. The high-risk group includes patients with risk 1 in 11 to 1 in 250, the indication is to perform either an invasive procedure or a NIPT test. The last group has critical risk where risk is higher than 1 in 10, it is indicated to perform an invasive procedure.

NIPT is based on the presence of fetal DNA in maternal blood. In 1997, it was discovered that there is the presence of cell-free DNA in maternal circulation, and that was the first step of a wide range of projects trying to study fetal DNA in maternal blood until NIPT was developed.

Cell free-DNA (cfDNA)

Cell-free DNA (cfDNA) is the DNA of a fetal organ released by apoptotic cells from the placenta, which is a permeable structure that allows the interchange of blood between the mother and the fetus. Cell free-DNA enters the maternal vessels and starts circulating in maternal blood, this DNA increases with gestation, and it can be detected since week 5 or 6 of pregnancy. Cell-free – DNA allows accessing fetal DNA in a non-invasive way. Moreover, it can’t be detected after delivery, there will be no free cell – DNA from previous miscarriages or pregnancies in maternal blood. cfDNA has a different size from maternal DNA and they also have different DNA signals, but with the available technologies, it’s not possible to separate them. It has been proved that the presence of fetal DNA increases at 10% at 10 weeks of pregnancy, and the other 90% will come from the mother at 10 weeks of pregnancy.

Noninvasive prenatal testing (NIPT) – a procedure

To perform the NIPT procedure, we need to collect blood in a steck Cell-free DNA tube, which prevents maternal cells from lysis and preserves fetal DNA. This blood sample has to be treated to isolate the DNA, and it has to be amplified. Afterward, DNA is evaluated and checked for nucleus or microdeletion syndromes. There are 2 methods to perform NIPT. The first 1 is Next Generation Sequencing (NGS), in this process, after DNA extraction, the whole genome is amplified and sequenced. The amplicons are aligned with a human sequence reference, and they are counted. To calculate the risk, a mathematical algorithm is used. The other method is the SNP-based NIPT. This is a technique based on the study of concrete regions of the genome that are highly polymorphic, which are the SNPs (Single-nucleotide polymorphism). These SNPs are used to create patterns among individuals to identify them. In this case, it is necessary to extract from both parents’ DNA, isolate it and amplify their SNPs. The same is done with the cell-free DNA tube. The parent SNPs pattern is compared to the cell-free DNA 1, and the copies of each chromosome are calculated through a mathematical algorithm that allows one to know the risk.

How does NIPT work? It is based on the concept of fatal fraction, which is the proportion of DNA that has origin from all the DNA found in the blood. The fetal fraction depends on BMI (Body Mass Index), gestational age (10w) and the presence of aneuploidies. At this point, the presence of aneuploidies is the clue for NIPT.

Aneuploidies and microdeletions cause a variation in fetal fraction. Having a genetically normal model, we can assume the fraction of maternal DNA is constant, and the variations are due to fetal number variation. this is used by a mathematical algorithm to calculate the risk. A fetal fraction can be a marker of either aneuploidies or copy number variations (CNVs), which are the ones that cause microdeletion syndromes.

It’s important to note that NIPT is a screening, but it is not a diagnostic test. That is why the results based on high-risk results should be confirmed by invasive procedures, while a low-risk result does not require any other procedure than a normal follow-up.

The performance of the test compared with other prenatal tests shows that NIPT has a diagnostic ability that equals the invasive test. This means that 99.9% of the results obtained by NIPT are confirmed with other tests. The main limitation of this test is that this is not a diagnostic test, and a positive result should always be confirmed by amniocentesis. To perform NIPT, there needs to be enough fetal material.

The first advantage of NIPT is that this is a non-invasive way to study fatal DNA. It is possible to approach DNA instead of phenotype, so it is possible to avoid the need to detect where phenotypes are not well described. It also has a high positive predicted value, which is the number of positive results that are real positives when used as a second test.

Take home messages

  • NIPT is a non-invasive screening test, which means that it is not a diagnostic tool
  • a high-risk result must be confirmed through an invasive procedure
  • NIPT is highly reliable when testing most common aneuploidies and CNVs
  • it’s a good complement to ultrasound monitoring
- Questions and Answers

Is it possible to use SNP based NIPT in an egg donation cycle, given the donation is anonymous?

This is a limitation of the SNPs (“snips”) based NIPT uses the maternal profile and the paternal profile to compare them as we know, the fetus would have a combination of maternal, and paternal profile. In this case, the maternal genetic load will be the egg donor’s genetic load, so it would be possible, but it’s a tricky field because we will have 3 different sources of DNA in the maternal’s blood. We will have the DNA from the maternal’s blood cells itself, and fatal DNA that will have half of its paternal pattern and half of the egg donor’s pattern. Theoretically, it can be done, but it’s a limitation of the snip based NIPT. It can be always done with NGS-based NIPT.

Why is sexual chromosomes sensibility lower than other chromosomes? 

It is mainly because the conditions due to sex chromosomes are less common in the population than those two other chromosomes. Sensitivity is calculated by the percentage of positives that are real positives. When we have a lot of positives that can be confirmed by an invasive procedure, we have a high sensitivity.

That is why the sensitivity is lower than with other trisomies such as 21, which causes down syndrome. Down syndrome is really common within other aneuploidies. We don’t have many sex chromosome aneuploidies. When we tried to do a theoretical sensitivity calculation, it had been higher than the real one because the real one is based on the actual cases we have. We don’t have that many cases, so that’s why it is slower than the sensitivity of other chromosomes.

When using NGS, will chromosomes be checked for aneuploidies or only sex chromosomes -13, 18, 21?

When using NGS, you can check every chromosome, such as 13, 18 and 21, which causes the most common aneuploidies related to autosomes, but you can also check the sex chromosomes. As I said, the sensitivity for each chromosome will vary because the sex chromosomes are not that common, but every chromosome can be checked.

How much fetal DNA is needed for NIPT?

That’s a tricky question, it’s difficult to know the quantity, we can’t say how many cells have suffered from lysis or have extracted the DNA, and they are the cell-free DNA we need. We can calculate is how much fatal fraction we need in the blood, and for the baby’s safety, it’s a 3.5% of fetal fraction. Also, cell-free DNA can be detected after five weeks of pregnancy, if I’m not wrong. It can be detected, but until the 9th week of pregnancy, we can’t calculate if we are having aneuploidy on the embryo or the differences in each chromosome are due to a low fetal fraction.

Does the NIPT test show gender?

The test can check the sexual chromosome, but the gender will be only revealed if the patient wants to know. We can test the sex chromosome and tell the patient that they are normal or abnormal. If the patient doesn’t want to know the gender, we cannot reveal it. We know the gender of the fetus because the mother is always XX, and so if we don’t have any copy of the Y chromosome, we assume that we are having a female XX, and if we have one copy of the Y chromosome, we assume that we are having a male XY.

When we are having twin pregnancies, it’s difficult to distinguish if the two of the fetus are males. When we have a Y copy, we can say that we have at least one male, but we cannot distinguish the second one, we cannot say if both of them are males or if you have one male and one female.

 Is NIPT a possible screening tool in twin pregnancies?

Yes, as I said, it’s possible, if you have a twin pregnancy or in your family, you have a twin brother, I would recommend looking for NIPT that uses NGS technologies because NGS is prepared for this kind of situations where we have a third source of DNA material. As I said, it’s possible, and the only tricky part is that we cannot distinguish the sex. Also, if we have a high risk on any of the chromosomes, even the sexual ones, we cannot distinguish, which of the fetus is the one that is affected, but you can do NIPT for sure.

What percentage do you detect of XXY?

I don’t know the numbers for the detection of XXY, so having two copies of the X chromosome and one copy of the Y chromosome. I don’t have the numbers, but as I said, the sensitivity of the test is high for the sex chromosomes, and the sensitivity is a bit lower than the ultrasounds, but it’s high. If you had more than 83% of the aneuploidies detected by NIPT, we can extrapolate that 83% (which is not a real number) of the XXY may be confirmed by an invasive procedure based on the data of the sex chromosomes.

Can the NIPT test be wrong?

NIPT can be wrong, but it happens very rarely. It can be comparable to invasive procedures because the positive predictive value is high, it’s 99.9%, which is even the maximum, but it always has to be confirmed by an invasive procedure. This is because we cannot separate the DNA coming from the mother and the DNA coming from the fetus.

We are doing maths calculations to know if the fetus has an aneuploidy or has a high risk of having an aneuploidy, and we must talk in these terms of the risk of having aneuploidies. As I said, every high-risk result on NIPT has to be confirmed, and that doesn’t mean that NIPT has a high positive predictive value, and I would recommend doing it before an invasive procedure.

Is NIPT the same test that is called the Harmony test in the UK?

I think the Harmony test is a commercial name for a NIPT, which is the technique.

What is SNP?

The snip is a Single-Nucleotide Polymorphism. In the DNA, we have a pair of bases that are named after letters: A, T, G, and C, normally, we have a sequence of these letters that are common in all humans, but sometimes in certain points of the DNA, we have changes that don’t affect our life, our development but are marking every individual.

I have certain letters of my DNA that are different from yours and my sister’s. These snips, these bases that are changed, can be used to create patterns that are different in every individual, this is used, for example, for fingerprinting. This is a really useful tool to find out if your DNA is actually from the mother or is a mix of the mother and the father.

Until when can you do the NIPT? Is the result more reliable if you wait longer?

It’s not more reliable, I think the reason for not waiting too much, it’s more based on not making the patient wait for the result. If you have passed the ninth week of pregnancy and still want to do NIPT, you can do it. You can also do a NIP if you are next to your delivery, but the reason why, and we are trying to do the screening earlier, even in the first trimester, is that we want to reduce the time of waiting for our first information about the fetus, so of course, you can do a NIPT any time during your pregnancy, and the result will not vary.

It is having been set up for the first trimester, but you can do it even after it. However, I don’t see the point of doing it after the first, the second trimester, where you have been doing echography, ultrasound, so maybe you have more information. If you go for a NIPT because your clinician has seen an abnormality in echography or an abnormal liver, level of hormones, of course, go for it.

If you have a normal pregnancy and are in your second or even third trimester, I don’t see the point of doing it and waiting that long. I would recommend doing it in the first semester.

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Blanca Rodríguez Estrada

Blanca Rodríguez Estrada

Blanca Rodríguez Estrada has a Bachelor’s Degree in Biology at Complutense University of Madrid (Spain) where she specialized in genetics. She coursed a Master’s Degree in Reproductive Medicine and Genetics at Miguel Hernández University, Alicante (Spain). She has been part of the iGLS team since July 2016, where she currently holds the position of Genetics Lab Manager. She is now working on her PhD based on her work at iGLS, which is the in-house Reproductive Genetics Laboratory of the IVF Life Group. iGLS offers ultimate solutions for infertility diagnosis and provides IVF Life Group clinics with advanced and integrated genetic services for reproductive medicine. She speaks English and Spanish.
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Caroline Kulczycka

Caroline Kulczycka

Caroline Kulczycka is managing MyIVFAnswers.com and has been hosting IVFWEBINARS dedicated to patients struggling with infertility since 2020. She's highly motivated and believes that educating patients so that they can make informed decisions is essential in their IVF journey. In the past, she has been working as an International Patient Coordinator, where she was helping and directing patients on their right path. She also worked in the tourism industry, and dealt with international customers on a daily basis, including working abroad. In her free time, you’ll find her travelling, biking, learning new things, or spending time outdoors.